SUMMARY Cryo-electron microscopy (cyro-EM) is undergoing extraordinary revolutions in technology, making it possible to image large protein assemblies and nucleic acid complexes in atomic detail. Critically however, the methods are mainly applicable to large assemblies (especially those that are symmetric), while smaller proteins (e.g. smaller than 50 kDa) are below current technological limits. Given that the average cellular protein is smaller than that, new molecular strategies are needed badly in order to fully realize the transformational potential of cryo-EM. It has long been recognized that smaller proteins of more typical size could be visualized by cryo-EM if they could be arrayed rigidly on a larger molecular scaffold to provide mass and higher contrast, and ideally in a way that would confer a high degree of symmetry. This goal has been largely elusive. Two key challenges have been (1) how to attach proteins to scaffolds in a rigid way so that important imaging advantages could be exploited, and (2) how to develop a modular system that would not require unpredictable molecular engineering and optimization and laborious EM testing for every new target molecule to be studied. This Technology Development proposal answers the challenge of developing cryo-EM scaffolds that are symmetric, modular, and rigid, by applying new methods from our group and collaborators for designing precisely defined and highly symmetric protein assemblies. Designed protein cages provide a symmetric core for external attachments. To these symmetric cores, we genetically fuse proteins known as DARPins, which have been developed as facile systems for binding specifically and rigidly to diverse proteins based on laboratory evolution of their loop sequences. Importantly, the connection between the DARPin and the designed symmetric core is made relatively rigid by using a continuous alpha helical fusion approach, an idea introduced earlier for designing novel protein cages. In preliminary work we evaluated one of the first-generation modular scaffold candidates by cryo- EM. When imaged by itself (cage core plus DARPin) the core is resolved to 3.1 and the DARPin is held rigidly enough to see at medium resolution (about 3.5 to 5.5 ). In new preliminary data the first scaffold candidate has been imaged with a first cargo protein, GFP, bound. There the DARPin is somewhat rigidified compared to the free form, while the bound GFP itself is visualized at 4.7 resolution overall, a new benchmark for cryo-EM of small proteins. These experiments lay out the most promising and most modular route so far for imaging small proteins, while also showing what further design steps are required to reach near-atomic resolution. This leading work will design, evaluate and perfect a novel set of cryo-EM scaffolds based on designed protein cages, with major impacts on structural biology and medicine.